The general protein secretory pathway: phylogenetic analyses leading to evolutionary conclusions

We have identified all homologues in the current databases of the ubiquitous protein constituents of the general secretory (Sec) pathway. These prokaryotic/eukaryotic proteins include (1) SecY/Sec61α, (2) SecE/Sec61γ, (3) SecG/Sec61β, (4) Ffh/SRP54 and (5) FtsY/SRP receptor subunit-α. Phylogenetic and sequence analyses lead to major conclusions concerning (1) the ubiquity of these proteins in living organisms, (2) the topological uniformity of some but not other Sec constituents, (3) the orthologous nature of almost all of them, (4) a total lack of paralogues in almost all organisms for which complete genome sequences are available, (5) the occurrence of two or even three paralogues in a few bacteria, plants, and yeast, depending on the Sec constituent, and (6) a tremendous degree of sequence divergence in bacteria compared with that in archaea or eukaryotes. The phylogenetic analyses lead to the conclusion that with a few possible exceptions, the five families of Sec constituents analyzed generally underwent sequence divergence in parallel but at different characteristic rates. The results provide evolutionary insights as well as guides for future functional studies. Because every organism with a fully sequenced genome exhibits at least one orthologue of each of these Sec proteins, we conclude that all living organisms have relied on the Sec system as their primary protein secretory/membrane insertion system. Because most prokaryotes and many eukaryotes encode within their genomes only one of each constituent, we also conclude that strong evolutionary pressure has minimized gene duplication events leading to the establishment of Sec paralogues. Finally, the sequence diversity of bacterial proteins as compared with their archaeal and eukaryotic counterparts is in agreement with the suggestion that bacteria were the evolutionary predecessors of archaea and eukaryotes.     

The iron/lead transporter superfamily of Fe/Pb2+ uptake systems

Oxidase-dependent ferrous iron uptake transporters of the OFeT family and lead uptake transporters of the PbrT family comprise the iron/lead transporter (ILT) superfamily (transporter classification No. 9.A.10). All sequenced homologues of the ILT superfamily were multiply aligned, and conserved motifs, including fully conserved acidic residues in putative transmembrane segments (TMSs) 1 and 4, previously implicated in heavy metal binding, were identified. Topological analyses confirmed the presence of 7 conserved TMSs in a 3 + 3 + 1 arrangement where the two 3 TMS elements are internally repeated. Phylogenetic analyses revealed the presence of several sequence divergent clusters of orthologous proteins that group roughly according to the phylogenes of the organisms of origin. The results serve to characterize and provide evolutionary insight into a novel superfamily of heavy metal uptake transporters.     

The general protein secretory pathway: phylogenetic analyses leading to evolutionary conclusions

We have identified all homologues in the current databases of the ubiquitous protein constituents of the general secretory (Sec) pathway. These prokaryotic/eukaryotic proteins include (1) SecY/Sec61alpha, (2) SecE/Sec61gamma, (3) SecG/Sec61beta, (4) Ffh/SRP54 and (5) FtsY/SRP receptor subunit-alpha. Phylogenetic and sequence analyses lead to major conclusions concerning (1) the ubiquity of these proteins in living organisms, (2) the topological uniformity of some but not other Sec constituents, (3) the orthologous nature of almost all of them, (4) a total lack of paralogues in almost all organisms for which complete genome sequences are available, (5) the occurrence of two or even three paralogues in a few bacteria, plants, and yeast, depending on the Sec constituent, and (6) a tremendous degree of sequence divergence in bacteria compared with that in archaea or eukaryotes. The phylogenetic analyses lead to the conclusion that with a few possible exceptions, the five families of Sec constituents analyzed generally underwent sequence divergence in parallel but at different characteristic rates. The results provide evolutionary insights as well as guides for future functional studies. Because every organism with a fully sequenced genome exhibits at least one orthologue of each of these Sec proteins, we conclude that all living organisms have relied on the Sec system as their primary protein secretory/membrane insertion system. Because most prokaryotes and many eukaryotes encode within their genomes only one of each constituent, we also conclude that strong evolutionary pressure has minimized gene duplication events leading to the establishment of Sec paralogues. Finally, the sequence diversity of bacterial proteins as compared with their archaeal and eukaryotic counterparts is in agreement with the suggestion that bacteria were the evolutionary predecessors of archaea and eukaryotes.     

A complex-centric view of protein network evolution

The recent availability of protein–protein interaction networks for several species makes it possible to study protein complexes in an evolutionary context. In this article, we present a novel network-based framework for reconstructing the evolutionary history of protein complexes. Our analysis is based on generalizing evolutionary measures for single proteins to the level of whole subnetworks, comprehensively considering a broad set of computationally derived complexes and accounting for both sequence and interaction changes. Specifically, we compute sets of orthologous complexes across species, and use these to derive evolutionary rate and age measures for protein complexes. We observe significant correlations between the evolutionary properties of a complex and those of its member proteins, suggesting that protein complexes form early in evolution and evolve as coherent units. Additionally, our approach enables us to directly quantify the extent to which gene duplication has played a role in the evolution of complexes. We find that about one quarter of the sets of orthologous complexes have originated from evolutionary cores of homodimers that underwent duplication and divergence, testifying to the important role of gene duplication in protein complex evolution.     

Evolution of sarcomeric myosin heavy chain genes: evidence from fish

Myosin heavy chain (MYH) is a major structural protein, integral to the function of sarcomeric muscles. We investigated both exon-intron organization and amino acid sequence of sarcomeric MYH genes to infer their evolutionary history in vertebrates. Our results were consistent with the hypothesis that a multigene family encoded MYH proteins in the ancestral chordate, one gene ancestral to human MYH16 and its homologues and another ancestral to all other vertebrate sarcomeric MYH genes. We identified teleost homologues of mammalian skeletal and cardiac MYH genes, indicating that the ancestors of those genes were present before the divergence of actinopterygians and sarcopterygians. Indeed, the ancestral skeletal genes probably duplicated at least once before the divergence of teleosts and tetrapods. Fish homologues of mammalian skeletal MYH are expressed in skeletal tissue and homologues of mammalian cardiac genes are expressed in the heart but, unlike mammals, there is overlap between these expression domains. Our analyses inferred two other ancestral vertebrate MYH genes, giving rise to human MYH14 and MYH15 and their homologues. Relative to the skeletal and cardiac genes, MYH14 and MYH15 homologues are characterized by evolution of intron position, differences in evolutionary rate between the functionally differentiated head and rod of the myosin protein, and possible evolution of function among vertebrate classes. Tandem duplication and gene conversion appear to have played major roles in the evolution of at least cardiac and skeletal MYH genes in fish. One outcome of this high level of concerted evolution is that different fish taxa have different suites of MYH genes, i.e., true orthologs do not exist.     

The I2 resistance gene homologues in Solanum have complex evolutionary patterns and are targeted by miRNAs

Background

Several resistance traits, including the I2 resistance against tomato fusarium wilt, were mapped to the long arm of chromosome 11 of Solanum. However, the structure and evolution of this locus remain poorly understood.

Results

Comparative analysis showed that the structure and evolutionary patterns of the I2 locus vary considerably between potato and tomato. The I2 homologues from different Solanaceae species usually do not have orthologous relationship, due to duplication, deletion and frequent sequence exchanges. At least 154 sequence exchanges were detected among 76 tomato I2 homologues, but sequence exchanges between I2 homologues in potato is less frequent. Previous study showed that I2 homologues in potato were targeted by miR482. However, our data showed that I2 homologues in tomato were targeted by miR6024 rather than miR482. Furthermore, miR6024 triggers phasiRNAs from I2 homologues in tomato. Sequence analysis showed that miR6024 was originated after the divergence of Solanaceae. We hypothesized that miR6024 and miR482 might have facilitated the expansion of the I2 family in Solanaceae species, since they can minimize their potential toxic effects by down-regulating their expression.

Conclusions

The I2 locus represents a most divergent resistance gene cluster in Solanum. Its high divergence was partly due to frequent sequence exchanges between homologues. We propose that the successful expansion of I2 homologues in Solanum was at least partially attributed to miRNA mediated regulation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-743) contains supplementary material, which is available to authorized users.     

Evolutionary significance of gene expression divergence

Recent large-scale studies of evolutionary changes in gene expression among mammalian species have led to the proposal that gene expression divergence may be neutral with respect to organismic fitness. Here, we employ a comparative analysis of mammalian gene sequence divergence and gene expression divergence to test the hypothesis that the evolution of gene expression is predominantly neutral. Two models of neutral gene expression evolution are considered: 1-purely neutral evolution (i.e., no selective constraint) of gene expression levels and patterns and 2-neutral evolution accompanied by selective constraint. With respect to purely neutral evolution, levels of change in gene expression between human-mouse orthologs are correlated with levels of gene sequence divergence that are determined largely by purifying selection. In contrast, evolutionary changes of tissue-specific gene expression profiles do not show such a correlation with sequence divergence. However, divergence of both gene expression levels and profiles are significantly lower for orthologous human-mouse gene pairs than for pairs of randomly chosen human and mouse genes. These data clearly point to the action of selective constraint on gene expression divergence and are inconsistent with the purely neutral model; however, there is likely to be a neutral component in evolution of gene expression, particularly, in tissues where the expression of a given gene is low and functionally irrelevant. The model of neutral evolution with selective constraint predicts a regular, clock-like accumulation of gene expression divergence. However, relative rate tests of the divergence among human-mouse-rat orthologous gene sets reveal clock-like evolution for gene sequence divergence, and to a lesser extent for gene expression level divergence, but not for the divergence of tissue-specific gene expression profiles. Taken together, these results indicate that gene expression divergence is subject to the effects of purifying selective constraint and suggest that it might also be substantially influenced by positive Darwinian selection.     

Selection-Driven Divergence After Gene Duplication in Arabidopsis thaliana

Gene duplications are one of the most important mechanisms for the origin of evolutionary novelties. Even though various models of the fate of duplicated genes have been established, current knowledge about the role of divergent selection after gene duplication is rather limited. In this study, we analyzed sequence divergence in response to neo- and subfunctionalization of segmentally duplicated genes in the genome of Arabidopsis thaliana. We compared the genomes of A. thaliana and the poplar Populus trichocarpa to identify orthologous pairs of genes and their corresponding inparalogs. Maximum-likelihood analyses of the nonsynonymous and synonymous substitution rate ratio [Formula: see text] of pairs of A. thaliana inparalogs were used to detect differences in the evolutionary rates of protein coding sequences. We analyzed 1,924 A. thaliana paralogous pairs and our results indicate that around 6.9% show divergent ω values between the lineages for a fraction of sites. We observe an enrichment of regulatory sequences, a reduced level of co-expression and an increased number of substitutions that can be attributed to positive selection based on an McDonald-Kreitman type of analysis. Taken together, these results show that selection after duplication contributes substantially to gene novelties and hence functional divergence in plants.     

Evolutionary conservation of expression profiles between human and mouse orthologous genes

Mouse models are often used to study human genes because it is believed that the expression and function are similar for the majority of orthologous genes between the two species. However, recent comparisons of microarray data from thousands of orthologous human and mouse genes suggested rapid evolution of gene expression profiles under minimal or no selective constraint. These findings appear to contradict non-array-based observations from many individual genes and imply the uselessness of mouse models for studying human genes. Because absolute levels of gene expression are not comparable between species when the data are generated by species-specific microarrays, use of relative mRNA abundance among tissues (RA) is preferred to that of absolute expression signals. We thus reanalyze human and mouse genome-wide gene expression data generated by oligonucleotide microarrays. We show that the mean correlation coefficient among expression profiles detected by different probe sets of the same gene is only 0.38 for humans and 0.28 for mice, indicating that current measures of expression divergence are flawed because the large estimation error (discrepancy in expression signal detected by different probe sets of the same gene) is mistakenly included in the between-species divergence. When this error is subtracted, 84% of human-mouse orthologous gene pairs show significantly lower expression divergence than that of random gene pairs. In contrast to a previous finding, but consistent with the common sense, expression profiles of orthologous tissues between species are more similar to each other than to those of nonorthologous tissues. Furthermore, the evolutionary rate of expression divergence and that of coding sequence divergence are found to be weakly, but significantly positively correlated, when RA and the Euclidean distance are used to measure expression-profile divergence. These results highlight the importance of proper consideration of various estimation errors in comparing the microarray data between species.     

Extensive sequence turnover of the signal peptides of members of the GDF/BMP family: exploring their evolutionary landscape

We show that the predicted signal peptide (SP) sequences of the secreted factors GDF9, BMP15 and AMH are well conserved in mammals but dramatic divergence is noticed for more distant orthologs. Interestingly, bioinformatic predictions show that the divergent protein segments do encode SPs. Thus, such SPs have undergone extensive sequence turnover with full preservation of functionality. This can be explained by a pervasive accumulation of neutral and compensatory mutations. An exploration of the potential evolutionary landscape of some SPs is presented. Some of these signal sequences highlight an apparent paradox: they are encoded, by definition, by orthologous DNA segments but they are, given their striking divergence, examples of what can be called functional convergence.     

Selection on rapidly evolving proteins in the Arabidopsis genome

Genes that have undergone positive or diversifying selection are likely to be associated with adaptive divergence between species. One indicator of adaptive selection at the molecular level is an excess of amino acid replacement fixed differences per replacement site relative to the number of synonymous fixed differences per synonymous site (omega = K(a)/K(s)). We used an evolutionary expressed sequence tag (EST) approach to estimate the distribution of omega among 304 orthologous loci between Arabidopsis thaliana and A. lyrata to identify genes potentially involved in the adaptive divergence between these two Brassicaceae species. We find that 14 of 304 genes (approximately 5%) have an estimated omega > 1 and are candidates for genes with increased selection intensities. Molecular population genetic analyses of 6 of these rapidly evolving protein loci indicate that, despite their high levels of between-species nonsynonymous divergence, these genes do not have elevated levels of intraspecific replacement polymorphisms compared to previously studied genes. A hierarchical Bayesian analysis of protein-coding region evolution within and between species also indicates that the selection intensities of these genes are elevated compared to previously studied A. thaliana nuclear loci.     

The ion transporter superfamily

We define a novel superfamily of secondary carriers specific for cationic and anionic compounds, which we have termed the ion transporter (IT) superfamily. Twelve recognized and functionally defined families constitute this superfamily. We provide statistical sequence analyses demonstrating that these families were in fact derived from a common ancestor. Further, we characterize the 12 families in terms of (1) the known substrates transported, (2) the modes of transport and energy coupling mechanisms used, (3) the family sizes (in numbers of sequenced protein members in the current NCBI database), (4) the organismal distributions of the members of each family, (5) the size ranges of the constituent proteins, (6) the predicted topologies of these proteins, and (7) the occurrence of non-homologous auxiliary proteins that may either facilitate or be required for transport. No member of the superfamily is known to function in a capacity other than transport. Proteins in several of the constituent families are shown to have arisen by tandem intragenic duplication events, but topological variation has resulted from a variety of dissimilar genetic fusion, splicing and insertional events. The evolutionary relationships between the members of each family are defined, leading to predictions of functionally relevant orthologous relationships. Some but not all of the families include functionally dissimilar paralogues that arose by early extragenic duplication events.     

Examination of protein sequence homologies: III. Ribosomal protein YS25 fromSaccharomyces cerevisiae and its counterparts fromSchizosaccharomyces pombe,rat liver,andEscherichia coli

Summary The sequences of the ribosomal proteins YS25, SP-S28, RL-S21, and Ec-S6, fromSaccharomyces cerevisiae, Schizosaccharomyces pombe, rat liver, andEscherichia coli, respectively, have been examined using a computer program that searches for homologous tertiary structures. Matrices of comparisons among the eukaryotic sequences show that they match each other sequentially without any internal gaps. The average values of the correlation coefficients obtained from the comparison matrices are higher for the first halves of the sequences than for the latter halves. This result suggests that the first halves of the sequences may represent a more important domain than the latter halves. The comparison matrices between the eukaryotic and bacterial sequences of ribosomal proteins, however, do not show sequentially arranged homology, though there are six well-matching segments arranged in different orders in the two types of sequences. This implies that the eukaryotic sequences of the ribosomal protein were reconstituted by two internal transpositions and six deletions of 4–12 residues each from the ancestral sequence during the divergence between bacterial and eukaryotic genes. These findings may give insight into structural and quantitative studies of evolutionary divergence between eukaryotes and prokaryotes.     

Ribonuclease A homologues of the zebrafish: polymorphism, crystal structures of two representatives and their evolutionary implications

The widespread and functionally varied members of the ribonuclease A (RNase A) superfamily provide an excellent opportunity to study evolutionary forces at work on a conserved protein scaffold. Representatives from the zebrafish are of particular interest as the evolutionary distance from non-ichthyic homologues is large. We conducted an exhaustive survey of available zebrafish DNA sequences and found significant polymorphism among its four known homologues. In an extension of previous nomenclature, the variants have been named RNases ZF-1a-c,-2a-d,-3a-e and-4. We present the first X-ray crystal structures of zebrafish ribonucleases, RNases ZF-1a and-3e at 1.35-and 1.85 Å resolution, respectively. Structure-based clustering with ten other ribonuclease structures indicates greatest similarity to mammalian angiogenins and amphibian ribonucleases, and supports the view that all present-day ribonucleases evolved from a progenitor with three disulphide bonds. In their details, the two structures are intriguing melting-pots of features present in ribonucleases from other vertebrate classes. Whereas in RNase ZF-1a the active site is obstructed by the C-terminal segment (as observed in angiogenin), in RNase ZF-3e the same region is open (as observed in more catalytically efficient homologues). The progenitor of present-day ribonucleases is more likely to have had an obstructive C terminus, and the relatively high similarity (late divergence) of RNases ZF-1 and-3 infers that the active site unblocking event has happened independently in different vertebrate lineages.     

Evolutionary sequence analysis of complete eukaryote genomes

Background  

Gene duplication and gene loss during the evolution of eukaryotes have hindered attempts to estimate phylogenies and divergence times of species. Although current methods that identify clusters of orthologous genes in complete genomes have helped to investigate gene function and gene content, they have not been optimized for evolutionary sequence analyses requiring strict orthology and complete gene matrices. Here we adopt a relatively simple and fast genome comparison approach designed to assemble orthologs for evolutionary analysis. Our approach identifies single-copy genes representing only species divergences (panorthologs) in order to minimize potential errors caused by gene duplication. We apply this approach to complete sets of proteins from published eukaryote genomes specifically for phylogeny and time estimation.     

Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels

Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels. Received: 30 September 1996 / Accepted: 15 November 1996     

Structural divergence is more extensive than sequence divergence for a family of intrinsically disordered proteins

The p53 transactivation domain (p53TAD) is an intrinsically disordered protein (IDP) domain that undergoes coupled folding and binding when interacting with partner proteins like the E3 ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70. The secondary structure and dynamics of six closely related mammalian homologues of p53TAD were investigated using nuclear magnetic resonance (NMR) spectroscopy. Differences in both transient secondary structure and backbone dynamics were observed for the homologues. Many of these differences were localized to the binding sites for MDM2 and RPA70. The amount of transient helical secondary structure observed for the MDM2 binding site was lower for the dog and mouse homologues, compared with human, and the amount of transient helical secondary structure observed for the RPA70 binding site was higher for guinea pig and rabbit, compared with human. Differences in the amount of transient helical secondary structure observed for the MDM2 binding site were directly related to amino acid substitutions occurring on the solvent exposed side of the amphipathic helix that forms during the p53TAD/MDM2 interaction. Differences in the amount of transient helical secondary structure were not as easily explained for the RPA70 binding site because of its extensive sequence divergence. Clustering analysis shows that the divergence in the transient secondary structure of the p53TAD homologues exceeds the amino acid sequence divergence. In contrast, strong correlations were observed between the backbone dynamics of the homologues and the sequence identity matrix, suggesting that the dynamic behavior of IDPs is a conserved evolutionary feature. Proteins 2013; 81:1686–1698. © 2013 Wiley Periodicals, Inc.